CN108998567B - Molecular marker primer and method for identifying amygdalus pedunculata pall, amygdalus mongolicus and prunus ulmaria - Google Patents
Molecular marker primer and method for identifying amygdalus pedunculata pall, amygdalus mongolicus and prunus ulmaria Download PDFInfo
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Abstract
A molecular marker primer and a method for identifying Amygdalus pedunculata, Prunus mongolica and Prunus ulmaria, wherein the molecular marker primer comprises a forward primer and a reverse primer; the sequence of the forward primer tKF: ACTCGAACCCGGAACTAG, the sequence of the reverse primer tKR: CGACCGATTTCTGCGTAT are provided. The identification method comprises the following steps: (1) collecting a leaf sample; (2) extracting total genomic DNA of the leaf sample; (3) amplifying target genes in the total genome DNA of the leaf sample in the step (2) by using molecular marker primers for identifying the amygdalus pedunculata pall, the amygdalus mongolicus and the prunus sibirica to obtain an amplification product; (4) purifying and sequencing the amplification product in the step (3); (5) and (4) carrying out alignment analysis on the sequencing result in the step (4). The base difference based on the trnK-UUUU intergenic region can be used as a molecular marker primer for identifying germplasm distinction of amygdalus pedunculata pall, amygdalus mongolicus and prunus sibirica, and the identification method is simple and easy to operate.
Description
Technical Field
The invention relates to the field of biotechnology. In particular to a molecular marker primer and a method for identifying amygdalus pedunculata, amygdalus mongolicus and prunus sibirica.
Background
The longstalck peaches (Prunus pedunculata Pall), the Prunus mongolica Maxim and the Prunus ulmaria (Prunus triloba Lindl) are special economic forest tree species in China, are naturally distributed in inner Mongolia, Liaoning, Hebei, Shanxi, Shaanxi, Gansu and other areas, have extremely strong cold and drought resistance characteristics, and are preferable tree species for ecological construction in semiarid desertification areas in China. The research shows that the oil content of the seeds of the longstalck peaches is about 56 percent, the oil content is higher than that of the Mongolian peaches and the elm plums, the oil is rich in unsaturated fatty acid (96 to 98 percent), the oil belongs to high oleic acid vegetable oil with oleic acid content higher than 70 percent, and the high oleic acid vegetable oil is equivalent to olive oil which is qualified as edible vegetable oil after queen, and is functional health-care oil which can be directly eaten. In 2013, the national health family planning committee brings the longstalck peaches into woody grain and oil tree species in China, and therefore, the longstalck peaches play an important role in guaranteeing the grain and oil safety and the ecological safety in China. In the amygdalus plants, amygdalus mongolica and amygdalus sibirica are perennial shrubs, are widely distributed, have extremely rich germplasm resources, and are often used as amygdalus mongolica and amygdalus sibirica in production and markets due to the similar growing environments of the amygdalus mongolica, the amygdalus sibirica and the amygdalus sibirica all have similar morphological characteristics and the like. The oil content and fatty acid component of the pseudo-Mongolian almond, the elm leaf and the longstalck almond are different, and the economic benefit of the longstalck almond cannot be replaced, so that the Mongolian almond, the elm leaf and the longstalck almond need to be accurately distinguished from each other when the Mongolian almond, the elm leaf and the longstalck almond are circulated in the market.
At present, the identification of the amygdalus pedunculata pall, the amygdalus mongolicus and the prunus sibirica is mainly carried out by traditional identification methods such as morphological character and microscopic identification, and the traditional identification method needs a growth period of 3-5 years and depends on skilled professional knowledge, thus being not beneficial to popularization.
In recent years, with the rapid development of molecular biology and bioinformatics, DNA barcode identification technology has become an effective tool for animal and plant identification because it is not affected by environment and morphological features, and has the advantages of simple and easy operation, high repeatability and stability, etc. In the plant DNA bar code, available genome DNA (ribonucleic acid) (nrDNA), chloroplast genome DNA (cpDNA) and mitochondrial genome DNA (mtDNA) are nucleated genome DNA (nrDNA), chloroplast genome DNA (cpDNA) and mitochondrial genome DNA (mtDNA), wherein the nrDNA has the fastest evolution rate, is a completely autonomous genetic system in a cell nucleus, has extremely complex structure and composition, forms a functional regulation network in each gene family, and has the problems of orthologous and paralogous and the like due to the multi-copy phenomenon of many genes. mtDNA is easy to generate gene rearrangement, duplication and loss, and the copy number of the mtDNA of the plant is low, so that the extraction and purification are difficult, and therefore, the application range of the nrDNA and the mtDNA in plant identification is limited. Compared with nrDNA and mtDNA, cpDNA has no more repetitive sequences and frequent rearrangement events, is not affected by genetic recombination, and has special significance for identifying complex species. Meanwhile, most chloroplast genomes belong to maternal inheritance, have relatively independent evolution routes and moderate evolution rate, and are suitable for identification and analysis of low classification orders such as genus and species. The plant chloroplast gene trnK-UUU has an important role in photosynthesis, and due to the functional limitation, the trnK-UU gene has a slow evolution speed, has small genetic variation at the genus and species level, has the characteristics of strong universality, easiness in amplification and the like, and is often selected as a DNA barcode for molecular identification in plant identification research. The method can be combined with a DNA rapid extraction and detection technology to search and establish a convenient identification method for 3 prunus plants.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a molecular identification primer and a method for amygdalus pedunculata pall, amygdalus mongolicus and prunus sibirica.
In order to solve the technical problems, the invention provides the following technical scheme:
a molecular marker primer for identifying amygdalus pedunculata pall, amygdalus mongolicus and prunus sibirica is a chloroplast genome.
The molecular marker primer for identifying the amygdalus pedunculata pall, the amygdalus mongolicus and the prunus sibirica comprises a forward primer and a reverse primer;
the sequence of the forward primer tKF (shown as SEQ ID NO: 1): ACTCGAACCCGGAACTAG the flow of the air in the air conditioner,
the sequence of the reverse primer tKR (shown as SEQ ID NO: 2): CGACCGATTTCTGCGTAT are provided.
A method for identifying amygdalus pedunculata, amygdalus mongolicus and prunus ulmaria comprises the following steps:
(1) collecting a leaf sample;
in the step (1), fresh, healthy and disease and pest-free leaf samples of amygdalus pedunculata pall, amygdalus mongolicus and prunus ulmaria in each region are collected to form an amygdalus pedunculata leaf sample group, an amygdalus mongolicus leaf sample group and a prunus ulmaria leaf sample group.
(2) Extracting total genomic DNA of the leaf sample;
in the step (2), the total genomic DNA of the amygdalus pedunculata leaf sample, the amygdalus mongolicus leaf sample and the elm leaf sample is extracted by a plant DNA rapid extraction kit.
(3) Amplifying target genes in the total genome DNA of the leaf sample in the step (2) by using molecular marker primers for identifying the amygdalus pedunculata pall, the amygdalus mongolicus and the prunus sibirica to obtain an amplification product;
in the step (3), identifying molecular marker primers of amygdalus pedunculata, amygdalus mongolicus and prunus sibirica, wherein the molecular marker primers comprise a forward primer and a reverse primer;
the sequence of the forward primer tKF (shown as SEQ ID NO: 1): ACTCGAACCCGGAACTAG the flow of the air in the air conditioner,
the sequence of the reverse primer tKR (shown as SEQ ID NO: 2): CGACCGATTTCTGCGTAT, respectively;
the target fragment sequence is as follows: trnK-UUUU.
In the step (3), the step (c),
the PCR amplification reaction system is as follows: a 20uL mixing system consisting of 0.2. mu.L of 2 XTaq Plus Master Mix, 1. mu.L of leaf sample total genomic DNA, 2. mu.L of 10 XBuffer, 0.5. mu.L of forward primer, 0.5. mu.L of reverse primer, 0.5. mu.L of dNTP, 0.5. mu.L of DMSO and 14.8. mu.L of sterile deionized water ddH 2O; the concentration of the total genomic DNA of the leaf sample is 10-20 ng.mu.L-1The concentration of the whole case primer and the reverse primer were 0.8. mu. mol. L-1The concentration of dNTPCR is 0.25 mol.L-1The mass content of DMSO is 10%;
the PCR reaction program is:
(P-1) pre-denaturation at 95 ℃ for 5min,
(P-2) denaturation at 95 ℃ for 30s,
(P-3) annealing at 52 ℃ for 30s,
(P-4) extension at 72 ℃ for 30s,
(P-5) the steps (P-1) to (P-4) are circulated 30 times,
(P-6) final extension at 72 ℃ for 5 min.
(4) Purifying and sequencing the amplification product in the step (3);
in the step (4), the PCR amplification product obtained in the step (3) is detected by a DNA analyzer.
(5) And (4) carrying out alignment analysis on the sequencing result in the step (4).
In the step (5), the GENEDOC software and the DNAMAN software are used for sequence comparison to obtain the base difference of the amygdalus pedunculata, the amygdalus mongolicus and the prunus ulmaria, and the species identification is carried out.
In the step (5), the basic group difference of the amygdalus pedunculata, the amygdalus mongolicus and the prunus ulmaria is used as a molecular marker for identifying germplasm, and the basic group difference of the amygdalus pedunculata, the amygdalus mongolicus and the prunus ulmaria is as follows:
(1) the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group have insertion of base A at the 75 th base position, and the amygdalus mongolica has deletion of the base A at the position;
(2) the amygdalus pedunculata leaf sample group had an insertion of base ATAGAAATAAATGCAAATTTTTT at the 246 th base position, and the amygdalus comfortis leaf sample group and the ulmus pumila leaf sample group had a deletion of base ATAGAAATAAATGCAAATTTTTT at this point;
(3) the base of the amygdalus pedunculata leaf sample group at the 292 th base position is G, and the base of the amygdalus mongolicus leaf sample group and the ulmus pumila leaf sample group at the position is T;
(4) the base of the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group at the 358 th base position is T, and the base of the amygdalus pedunculata leaf sample group at this position is C;
(5) the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group at the 429 base position have a base A, and the amygdalus pedunculata leaf sample group has a base G at this position.
Advantageous effects
1. The total genome DNA of the amygdalus pedunculata pall leaf, the amygdalus mongolica leaf and the amygdalus ulmaria leaf sample is amplified by using a forward primer tKF and a reverse primer tKR, and the amplified chloroplast DNA trnK-UUUU gene interval short segment sequence comparison result shows that 5 base variation sites exist among the amygdalus pedunculata pall, the amygdalus mongolica and the amygdalus ulmaria leaf sequence, wherein 3 conversion sites and 2 insertion/deletion sites are respectively formed. The amplification results of the longstalck peaches in the population are consistent, the amplification results of the Mongolian peaches in the population are consistent, and the amplification results of the elm plum in the population are also consistent and show high consistency. The method has stable result and strong repeatability, and ensures extremely high accuracy, so that the forward primer tKF and the reverse primer tKR can be used as molecular marker primers for identifying germplasm differences of amygdalus pedunculata pall, amygdalus mongolicus and prunus sibirica based on the base difference of the trnK-UUU intergenic spacer region, and the identification method is simple and easy to operate.
2. The chloroplast DNA barcode is more suitable for molecular identification of various plants because the chloroplast gene copy number is more than that of the nuclear gene, and the chloroplast gene is less susceptible to DNA degradation than the nuclear gene.
3. The traditional morphological characteristic detection method can be finished by observing and identifying plant leaves, flowers, fruits and the like for a long time (3-5 years), and is easily influenced by environment and human, only one leaf is needed to quickly identify a sample, the detection speed and efficiency are greatly improved, and a quick and simple detection system is established for the method.
Drawings
FIG. 1 is an electrophoresis detection diagram for detecting DNA quality maps of extracted longstalck peach leaf samples, Mongolian almond leaf samples and elm leaf samples by agarose gel electrophoresis, (1-5 are longstalck peach leaf samples, 6-10 are Mongolian almond leaf samples, and 11-15 are elm leaf samples);
FIG. 2 is an electrophoresis detection diagram for detecting trnK-UUUU gene specific PCR amplification products of a longstalck leaf sample, a Mongolian almond leaf sample and an elm leaf sample by agarose gel electrophoresis, (1-5 are the longstalck leaf samples, 6-10 are the Mongolian almond leaf samples, and 11-15 are the elm leaf samples);
FIG. 3 shows the left half of the identification method for the longstalck leaf sample, the Mongolian almond leaf sample and the Japanese apricot leaf sample (1-5 for the longstalck leaf sample, 6-10 for the Mongolian almond leaf sample, and 11-15 for the Japanese apricot leaf sample);
fig. 4 right half of the schematic diagram of the methods for identifying the amygdalus pedunculata leaf sample, the amygdalus mongolica leaf sample and the amygdalus comani leaf sample (1-5 are amygdalus pedunculata leaf samples, 6-10 are amygdalus comnnis leaf samples, and 11-15 are amygdalus comani leaf samples).
Detailed Description
Apparatus and materials
PCR apparatus (Eppendorf, model 5332), cryocentrifuge (Eppendorf, model 5810R), micropipette (Eppendorf), electrophoresis system (Hexay instruments, Beijing, model DYY-12), gel imaging analyzer (BIO-RADChemidoc XRS), ABI 3500XL (Applied Biosystems, USA) DNA analyzer.
Plant DNA rapid extraction kit (Beijing Tiangen company), TAE buffer solution, agarose (Promega company), Goldview (Beijing Sorley Bay science and technology Co., Ltd.), DNA Taq polymerase (Takara company), chloroform, and absolute ethanol are domestic analytical purifications.
Example 1
1. Collection of leaf samples
The samples are identified and collected by an improved variety breeding center of inner Mongolian forest trees, and are collected from fresh, tender, healthy and pest-free leaf samples of amygdalus pedunculata pall, amygdalus mongolicus and Prunus ulmarius in different producing areas respectively by 5 parts. As described in table 1.
TABLE 1 leaf samples and sources of Amygdalus pedunculata, Prunus mongolica and Prunus ulmaria
2. Extraction of leaf sample Total genomic DNA
The total genome DNA of the leaf sample is extracted by the plant DNA rapid extraction kit, the quality of the leaf sample is detected based on 1.2 percent agarose gel electrophoresis, the result shows that as shown in figure 1, the brightness, the uniformity, the spot sample port light spot and the like of an electrophoresis strip are well displayed, the purity of the sample is high, no pollution is caused, and the plant DNA rapid extraction kit can be directly used for subsequent molecular biology experiments.
PCR amplification of target Gene sequences in Total genomic DNA of leaf samples
Amplifying a target fragment sequence trnK-UUU sequence by using a specific primer, namely a forward primer tKF and a reverse primer tKR;
the sequence of the forward primer tKF (shown as SEQ ID NO: 1): ACTCGAACCCGGAACTAG the flow of the air in the air conditioner,
the sequence of the reverse primer tKR (shown as SEQ ID NO: 2): CGACCGATTTCTGCGTAT are provided.
The PCR amplification reaction system is as follows: a 20uL mixing system consisting of 0.2. mu.L of 2 XTaq Plus Master Mix, 1. mu.L of leaf sample total genomic DNA, 2. mu.L of 10 XBuffer, 0.5. mu.L of forward primer, 0.5. mu.L of reverse primer, 0.5. mu.L of dNTP, 0.5. mu.L of DMSO and 14.8. mu.L of sterile deionized water ddH 2O; the concentration of the total genomic DNA of the leaf sample is 10-20 ng.mu.L-1The concentration of the whole case primer and the reverse primer were 0.8. mu. mol. L-1The concentration of dNTPCR is 0.25 mol.L-1The mass content of DMSO is 10%;
the PCR reaction program is:
(P-1) pre-denaturation at 95 ℃ for 5min,
(P-2) denaturation at 95 ℃ for 30s,
(P-3) annealing at 52 ℃ for 30s,
(P-4) extension at 72 ℃ for 30s,
(P-5) the steps (P-1) to (P-4) are circulated 30 times,
(P-6) final extension at 72 ℃ for 5 min.
4. Purifying and sequencing the amplification product
PCR amplification products of 15 leaf samples were detected using an ABI 3500XL (Applied Biosystems, USA) DNA analyzer.
5. Comparing and analyzing sequencing results
And (3) comparing the trnK-UUUU gene interval fragment sequences of 15 leaf samples obtained by sequencing by using GENEDOC and DNAMAN software to obtain specific INDELs and SNPs of amygdalus pedunculata, amygdalus mongolicus and amygdalus sibirica, and identifying the species.
Example 2 analysis of results
After the PCR product is purified, performing bidirectional sequencing, and comparing sequences of the sample group of the amygdalus pedunculata pall, the sample group of the amygdalus mongolicus pall and the sample group of the elm leaf, wherein the comparison result of the sample group of the amygdalus pedunculata pall, the sample group of the amygdalus mongolicus pall and the sample group of the elm leaf in the fragment sequence of the chloroplast gene trnK-UUU is as follows:
amygdalus pedunculata Roxb:
ACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTGATTAGAAAATTTTAAATAAAATAGGGAAAAA(A)+CCCCTCCCCAAACCGTGCTTGCATTTTTCATTGCACACGGCTTTCCCTATGTATACATCTAAAATTCAGTCCCTTCCCTATACACGACTCTAAGAAAGTTGAATACTCAGTTGATCGACCCTCAATCTTACTGTATGAACATTTCATAATAGAAATAAATGCAAATTTTTT(ATAGAAATAAATGCAAATTTTTT)+GTTATCTCTTCATTATTTAAAGAGAATTCCATTTCTACGATCGCATAACCAATTATTCATAATTGATTAGATCATTGATGCAAAAAATATCCAAATACCAAATCCGACCTCTATAAAATTTCTTAAAAGTAAAAGTATAAGAAGCTCTTGGGAAGACCAAAGAAAGAATCTGTTCTTCTTCCGTAAAGAATTCTTCCAATAATTTAGAACCTAATCTTTTCAAAAAAGTTCGTACAGTACTTTTGTGTTTACGAGCCAAAGTTTTAAGACAAGAAAGTCGAAGTATATATTTTATTCGATACAAACTCTTTTTTCTTGAGGATCCGCTGTAATAATGAGAAAGATTTCTGCATATACGCAGAAATCGGTCG
2, mongolian almond:
ACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTGATTAGAAAATTTTAAATAAAATAGGGAAAAA-CCCCTCCCCAAACCGTGCTTGCATTTTTCATTGCACACGGCTTTCCCTATGTATACATCTAAAATTCAGTCCCTTCCCTATACACGACTCTAAGAAAGTTGAATACTCAGTTGATCGACCCTCAATCTTACTGTATGAACATTTCATAATAGAAATAAATGCAAATTTTTT-----------------------GTTATCTCTTCATTATTTAAAGATAATTCCATTTCTACGATCGCATAACCAATTATTCATAATTGATTAGATCATTGATGCAAAAAATACCCAAATACCAAATCCGACCTCTATAAAATTTCTTAAAAGTAAAAGTATAAGAAGCTCTTGGGAAGACCAAGGAAAGAATCTGTTCTTCTTCCGTAAAGAATTCTTCCAATAATTTAGAACCTAATCTTTTCAAAAAAGTTCGTACAGTACTTTTGTGTTTACGAGCCAAAGTTTTAAGACAAGAAAGTCGAAGTATATATTTTATTCGATACAAACTCTTTTTTCTTGAGGATCCGCTGTAATAATGAGAAAGATTTCTGCATATACGCAGAAATCGGTCG
3, plum fruit elm:
ACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTGATTAGAAAATTTTAAATAAAATAGGGAAAAAACCCCTCCCCAAACCGTGCTTGCATTTTTCATTGCACACGGCTTTCCCTATGTATACATCTAAAATTCAGTCCCTTCCCTATACACGACTCTAAGAAAGTTGAATACTCAGTTGATCGACCCTCAATCTTACTGTATGAACATTTCATAATAGAAATAAATGCAAATTTTTT-----------------------GTTATCTCTTCATTATTTAAAGATAATTCCATTTCTACGATCGCATAACCAATTATTCATAATTGATTAGATCATTGATGCAAAAAATATCCAAATACCAAATCCGACCTCTATAAAATTTCTTAAAAGTAAAAGTATAAGAAGCTCTTGGGAAGACCAAAGAAAGAATCTGTTCTTCTTCCGTAAAGAATTCTTCCAATAATTTAGAACCTAATCTTTTCAAAAAAGTTCGTACAGTACTTTTGTGTTTACGAGCCAAAGTTTTAAGACAAGAAAGTCGAAGTATATATTTTATTCGATACAAACTCTTTTTTCTTGAGGATCCGCTGTAATAATGAGAAAGATTTCTGCATATACGCAGAAATCGGTCG
wherein, "()+"denotes insertion of a base sequence; "-" represents a deletion of the base sequence; the underlined "_" bases and corresponding bases are transition-specific variations.
The sequencing results of trnK-UUU gene fragment sequences of the amygdalus pedunculata leaf sample group, the amygdalus mongolicus leaf sample group and the elm leaf sample group show that 5 variation sites exist among the trnK-UUU gene fragment sequences, 3 conversions exist, 2 insertions/deletions exist, and three types can be effectively identified:
(1) the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group have insertion of a base A at the 75 th base position, and the amygdalus mongolicus has deletion of the base A at the position;
(2) the amygdalus pedunculata leaf sample group had an insertion of base ATAGAAATAAATGCAAATTTTTT at the 246 th base position, and the amygdalus comfortis leaf sample group and the ulmus pumila leaf sample group had a deletion of base ATAGAAATAAATGCAAATTTTTT at this point;
(3) the base of the amygdalus pedunculata leaf sample group at the 292 th base position is G, and the base of the amygdalus mongolicus leaf sample group and the ulmus pumila leaf sample group at the position is T;
(4) the base of the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group at the 358 th base position is T, and the base of the amygdalus pedunculata leaf sample group at this position is C;
(5) the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group at the 429 base position have a base A, and the amygdalus pedunculata leaf sample group has a base G at this position.
Sequencing results show that 5 individuals of the Amygdalus pedunculata leaf sample group have consistent amplification results, and 5 individuals of the Amygdalus pedunculata leaf sample group also have consistent amplification results, and the high consistency is shown, the results are stable and strong in repeatability, and extremely high accuracy is guaranteed.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
<110> university of inner Mongolia agriculture
<120> molecular marker primer and method for identifying amygdalus pedunculata pall, amygdalus mongolica and prunus ulmaria
<150> 2018105050166
<151> 2018-05-24
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
actcgaaccc ggaactag 18
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgaccgattt ctgcgtat 18
Claims (5)
1. A method for identifying amygdalus pedunculata pall, amygdalus mongolicus pall and prunus sibirica is characterized by comprising the following steps:
(1) collecting a leaf sample;
(2) extracting total genomic DNA of the leaf sample;
(3) amplifying target genes in the total genome DNA of the leaf sample in the step (2) by using molecular marker primers for identifying the amygdalus pedunculata pall, the amygdalus mongolicus and the prunus sibirica to obtain an amplification product;
molecular marker primers for identifying amygdalus pedunculata, amygdalus mongolicus and prunus ulmaria comprise a forward primer and a reverse primer;
the sequence of the forward primer tKF: ACTCGAACCCGGAACTAG the flow of the air in the air conditioner,
the sequence of the reverse primer tKR: CGACCGATTTCTGCGTAT, respectively;
the target genes are: trnK-UUUU;
(4) purifying and sequencing the amplification product in the step (3);
(5) comparing and analyzing the sequencing result in the step (4),
performing sequence comparison by using GENEDOC and DNAMAN software to obtain base difference of Amygdalus pedunculatus, Prunus mongolica and Prunus ulmarius, and performing species identification;
the basic group differences of the amygdalus pedunculata pall, the amygdalus mongolicus and the prunus ulmaria are used as molecular markers for identifying germplasm, and the basic group differences of the amygdalus pedunculata pall, the amygdalus mongolicus and the prunus ulmaria are as follows:
(1) the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group have insertion of base A at the 75 th base position of the amplification product, and the amygdalus mongolicus has deletion of base A;
(2) the amygdalus pedunculata leaf sample group had an insertion of base ATAGAAATAAATGCAAATTTTTT at the 246 th base position of the amplification product, and the amygdalus comfrey leaf sample group and the ulmus pumila leaf sample group had a deletion of base ATAGAAATAAATGCAAATTTTTT at this point;
(3) the base of the amygdalus pedunculata leaf sample group at the 292 th base position of the amplification product is G, and the base of the amygdalus mongolicus leaf sample group and the ulmus pumila leaf sample group at this point is T;
(4) the base of the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group at the 358 th base position of the amplification product is T, and the base of the amygdalus pedunculata leaf sample group at this point is C;
(5) the base of the amygdalus pedunculata leaf sample group and the amygdalus pedunculata leaf sample group at the 429 th base position of the amplification product is A, and the base of the amygdalus pedunculata leaf sample group at this position is G.
2. The method of claim 1, wherein in step (1), samples of fresh, healthy and pest-free leaves of amygdalus pedunculata, amygdalus mongolicus and amygdalus sibiricus in each area are collected to form a sample group of amygdalus pedunculata leaves, a sample group of amygdalus mongolicus leaves and an sample group of amygdalus sibiricus leaves.
3. The method of discriminating amygdalus pedunculata, amygdalus mongolicus, and amygdalus sibiricus according to claim 2, wherein in the step (2), the total genomic DNA of the amygdalus pedunculata leaf sample, the amygdalus mongolicus leaf sample, and the amygdalus sibiricus leaf sample is extracted by a plant DNA rapid extraction kit.
4. The method of discriminating Amygdalus pedunculata, Prunus mongolica and Prunus ulmarius as claimed in claim 1, wherein, in the step (3),
the PCR amplification reaction system is as follows: a20 uL mixing system was included containing 0.2. mu.L of 2 XTaq Plus Master Mix, 1. mu.L of leaf sample total genomic DNA, 2. mu.L of 10 XBuffer, 0.5. mu.L of forward primer, 0.5. mu.L of reverse primer, 0.5. mu.L of dNTP, 0.5. mu.L of DMSO and 14.8. mu.L of sterile deionized water ddH2O; said leafThe concentration of the total genomic DNA of the chip sample is 10-20 ng.mu.L-1The concentrations of the forward primer and the reverse primer were 0.8. mu. mol. multidot.L-1The concentration of dNTP is 0.25 mol.L-1The mass content of DMSO is 10%;
the PCR reaction program is:
(P-1) pre-denaturation at 95 ℃ for 5min,
(P-2) denaturation at 95 ℃ for 30s,
(P-3) annealing at 52 ℃ for 30s,
(P-4) extension at 72 ℃ for 30s,
(P-5) the steps (P-1) to (P-4) are circulated 30 times,
(P-6) final extension at 72 ℃ for 5 min.
5. The method for discriminating Amygdalus pedunculata, Amygdalus mongolicus and Prunus ulmarius according to claim 4, wherein in the step (4), the PCR amplification product obtained in the step (3) is detected by a DNA analyzer.
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